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Over the last seven decades, γ-aminobutyric acid (GABA) has attracted great attention from scientists for its ubiquity in plants, animals and microorganisms and for its physiological implications as a signaling molecule involved in multiple pathways and processes. Recently, the food and pharmaceutical industries have also shown significantly increased interest in GABA, because of its great potential benefits for human health and the consumer demand for health-promoting functional compounds, resulting in the release of a plethora of GABA-enriched products. Nevertheless, many crop species accumulate appreciable GABA levels in their edible parts and could help to meet the daily recommended intake of GABA for promoting positive health effects. Therefore, plant breeders are devoting much effort into breeding elite varieties with improved GABA contents. In this regard, tomato ( Solanum lycopersicum ), the most produced and consumed vegetable worldwide and a fruit-bearing model crop, has received much consideration for its accumulation of remarkable GABA levels. Although many different strategies have been implemented, from classical crossbreeding to induced mutagenesis, new plant breeding techniques (NPBTs) have achieved the best GABA accumulation results in red ripe tomato fruits along with shedding light on GABA metabolism and gene functions. In this review, we summarize, analyze and compare all the studies that have substantially contributed to tomato GABA breeding with further discussion and proposals regarding the most recent NPBTs that could bring this process to the next level of precision and efficiency. This document also provides guidelines with which researchers of other crops might take advantage of the progress achieved in tomato for more efficient GABA breeding programs.

Native/endogenous promoters have several fundamental limitations in terms of their size, Cis-elements distribution/patterning, and mode of induction, which is ultimately reflected in their insufficient transcriptional activity. Several customized synthetic promoters were designed and tested in plants during the past decade to circumvent such constraints. Such synthetic promoters have a built-in capacity to drive the expression of the foreign genes at their maximum amplitude in plant orthologous systems. The basic structure and function of the promoter has been discussed in this review, with emphasis on the role of the Cis-element in regulating gene expression. In addition to this, the necessity of synthetic promoters in the arena of plant biology has been highlighted. This review also provides explicit information on the two major approaches for developing plant-based synthetic promoters: the conventional approach (by utilizing the basic knowledge of promoter structure and Cis-trans interaction) and the advancement in gene editing technology. The success of plant genetic manipulation relies on the promoter efficiency and the expression level of the transgene. Therefore, advancements in the field of synthetic promoters has enormous potential in genetic engineering-mediated crop improvement.

Advances in systematic computational biology and rapid elucidation of synergistic interplay between cis and trans factors governing transcriptional control have facilitated functional annotation of gene networks. The generation of data through deconstructive, reconstructive and database assisted promoter studies, and its integration to principles of synthetic engineering has started an era of designer promoters. Exploration of natural promoter architecture and the concept of cis engineering have not only enabled fine tuning of single or multiple transgene expression in response to perturbations in the chemical, physiological and environmental stimuli but also provided researchers with a unique answer to various problems in crop improvement in the form of bidirectional promoters.

Meristem function underlies organogenesis and yield potential in crop species, and its regulation depends on the crosstalk of genetic and hormonal networks that balance stem-cell niche maintenance and differentiation. During the shoot apical meristem (SAM) transition, developmental reprogramming shifts the meristem from a vegetative to a reproductive state, referred to as inflorescence meristem (IM). Major regulatory events in this transition include the cytokinin–gibberellin crosstalk, that regulate the expression of the CLAVATA/WUSCHEL ( CLV/WUS ) negative feedback loop and key transcription factor families like KNOTTED-LIKE HOMEOBOX ( KNOX ) and SHOOT MERISTEMLESS ( STM ). Despite the basic principles of apical meristem differentiation are well-described nowadays, major phenotypic bottlenecks were reached in major staple crops during the artificial selection process, known as domestication, leading to a final reduction in total crop yield. This review aims to describe the key processes and genes that play a role in this transition and how they can be artificially targeted to overcome these limitations. Major bioengineering approaches are covered, ranging from classical random mutagenesis with chemicals like ethyl methanesulfonate (EMS) to targeted genome editing using diverse Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins (CRISPR/Cas) systems. Finally, emerging strategies such as epibreeding are considered as promising tools to achieve precise, reversible modulation of meristem activity and to unlock new routes for crop yield enhancement.

Main conclusion This paper reviews the importance, prospective and development of synthetic promoters reported in planta. A review of the synthetic promoters developed in planta would help researchers utilize the available resources and design new promoters to benefit fundamental research and agricultural applications. The demand for promoters for the improvement and application of transgenic techniques in research and agricultural production is increasing. Native/naturally occurring promoters have some limitations in terms of their induction conditions, transcription efficiency and size. The strength and specificity of native promoter can be tailored by manipulating its 'cis-architecture' by the use of several recombinant DNA technologies. Newly derived chimeric promoters with specific attributes are emerging as an efficient tool for plant molecular biology. In the last three decades, synthetic promoters have been used to regulate plant gene expression. To better understand synthetic promoters, in this article, we reviewed promoter structure, the scope of cis-engineering, strategies for their development, their importance in plant biology and the total number of such promoters (188) developed in planta to date; we then categorized them under different functional regimes as biotic stress-inducible, abiotic stress-inducible, light-responsive, chemical-inducible, hormone-inducible, constitutive and tissue-specific. Furthermore, we identified a set of 36 synthetic promoters that control multiple types of expression in planta. Additionally, we illustrated the differences between native and synthetic promoters and among different synthetic promoter in each group, especially in terms of efficiency and induction conditions. As a prospective of this review, the use of ideal synthetic promoters is one of the prime requirements for generating transgenic plants suitable for promoting sustainable agriculture and plant molecular farming.

Phenomics characterization of Erwinia spp. strain LJJL01 unveils the inherently robust metabolic pathways to utilize diverse substrates derived from biomass and plastics.The strain exhibits high chemical tolerance to biomass- and plastic-derived chemicals, including aldehydes.Advanced synthetic biology tools have been established for genetic programming of the strain.Metabolic pathways of the strain were rewired to produce non-native cis,cis-muconate and polyhydroxybutyrate from waste feedstock.The strain can be utilized as a host for biotransformations of both industrial and environmental interest. Nonmodel microbial hosts can efficiently biotransform unconventional organic feedstocks into advanced bioproducts, leveraging their superior metabolic capabilities and resilience to process-relevant physicochemical conditions. In this study, we domesticated Erwinia spp. strain. LJJL01 (Er LJJL01) as a potent microbial chassis to advance the emerging biorefinery strategy for the valorization of lignin-rich biomass and plastic, thereby enabling the circular economy. The strain exhibits remarkable chemical tolerance and can biofunnel various substrates, including sugars, acids, polyols, and aromatics, in minimal salt media to produce native fine chemicals such as acetoin, 2,3-butanediol, and lactic acid. As a proof of concept, engineering this strain with advanced genetic tools enables the production of tailored high-value chemicals, such as cis,cis-muconate from plastic-derived terephthalate and polyhydroxybutyrate from lignocellulosic hydrolysate. We established Er LJJL01 as a potent microbial chassis for the green synthesis of bioproducts from unconventional organic feedstocks. [Display omitted] Developing robust microbial chassis for bioproducts plays a key role in creating sustainable alternatives to fossil fuel-based synthetic chemical manufacturing processes. In this study, we demonstrated Erwinia spp. strain LJJL01 as a robust nonmodel microbial chassis for biomass and plastic waste valorization. As a proof of concept, we engineered Erwinia spp. strain LJJL01 by leveraging its inherent metabolic versatility and physiological robustness and harnessing the developed genetic toolkit to bioconvert plastic-derived monomers and biomass-derived heterogeneous organic carbon into performance-advanced chemicals. This proof-of-concept study reaches a technology readiness level of 3. However, the yield, titer, and rate of these processes still need improvement to achieve commercial viability. In-depth multiomics-guided approaches can be applied to deregulate competitive pathways, generate genome-reduced strains to diminish traits useless in a bioreactor (e.g., prophages and flagella), uncover genetic traits for designing genetic circuits to modulate metabolic pathways to enhance tailored product formation, and create a robust metabolic model for the in silico design of synthetic pathways. Although Erwinia spp. strain LJJL01 lacks any genomic signature that would raise concerns about its potential hazards, the strain needs to be certified by the US FDA as a host–vector system at a safety level 1 to be used in the lowest risk-level environment. Prior to industrial deployment, engineered Erwinia spp. strain LJJL01 requires managing risks from containment through the development of kill switches and genetic firewalls, as well as genetic barcode-based tracing in the environment. Implementing programmable flocculation and biomass recovery, conditional lysis, and other operational enhancements will further improve the strain and aid in a cost-effective downstream process. Enhancing the chemical resilience of the strain further via tolerance adaptive laboratory evolution broadens the applicability of the strain to form diverse bioproduct(s) under chemical stress and enables its use in industrial organic waste stream valorization (e.g., thermochemical aqueous waste). Erwinia spp. strain LJJL01 is a metabolically versatile and robust microbe capable of valorizing unconventional feedstocks, such as biomass and plastic, into high-value platform chemicals. We engineered this strain to demonstrate its potential for producing bioplastic precursors from waste feedstocks and showcased its applicability in biomanufacturing.

Summary Natural rubber (NR) is a nonfungible and valuable biopolymer, used to manufacture ~50 000 rubber products, including tires and medical gloves. Current production of NR is derived entirely from the para rubber tree (Hevea brasiliensis). The increasing demand for NR, coupled with limitations and vulnerability of H. brasiliensis production systems, has induced increasing interest among scientists and companies in potential alternative NR crops. Genetic/metabolic pathway engineering approaches, to generate NR‐enriched genotypes of alternative NR plants, are of great importance. However, although our knowledge of rubber biochemistry has significantly advanced, our current understanding of NR biosynthesis, the biosynthetic machinery and the molecular mechanisms involved remains incomplete. Two spatially separated metabolic pathways provide precursors for NR biosynthesis in plants and their genes and enzymes/complexes are quite well understood. In contrast, understanding of the proteins and genes involved in the final step(s)—the synthesis of the high molecular weight rubber polymer itself—is only now beginning to emerge. In this review, we provide a critical evaluation of recent research developments in NR biosynthesis, in vitro reconstitution, and the genetic and metabolic pathway engineering advances intended to improve NR content in plants, including H. brasiliensis, two other prospective alternative rubber crops, namely the rubber dandelion and guayule, and model species, such as lettuce. We describe a new model of the rubber transferase complex, which integrates these developments. In addition, we highlight the current challenges in NR biosynthesis research and future perspectives on metabolic pathway engineering of NR to speed alternative rubber crop commercial development.

Restoring vision in inherited retinal degenerations remains an unmet medical need. In mice exhibiting a genetically engineered block of the visual cycle, vision was recently successfully restored by oral administration of 9-cis-retinyl acetate (QLT091001). Safety and visual outcomes of a once-daily oral dose of 40 mg/m2/day QLT091001 for 7 consecutive days was investigated in an international, multi-center, open-label, proof-of-concept study in 18 patients with RPE65- or LRAT-related retinitis pigmentosa. Eight of 18 patients (44%) showed a ≥20% increase and 4 of 18 (22%) showed a ≥40% increase in functional retinal area determined from Goldmann visual fields; 12 (67%) and 5 (28%) of 18 patients showed a ≥5 and ≥10 ETDRS letter score increase of visual acuity, respectively, in one or both eyes at two or more visits within 2 months of treatment. In two patients who underwent fMRI, a significant positive response was measured to stimuli of medium contrast, moving, pattern targets in both left and right hemispheres of the occipital cortex. There were no serious adverse events. Treatment-related adverse events were transient and the most common included headache, photophobia, nausea, vomiting, and minor biochemical abnormalities. Measuring the outer segment length of the photoreceptor layer with high-definition optical coherence tomography was highly predictive of treatment responses with responders having a significantly larger baseline outer segment thickness (11.7 ± 4.8 μm, mean ± 95% CI) than non-responders (3.5 ± 1.2 μm). This structure-function relationship suggests that treatment with QLT091001 is more likely to be efficacious if there is sufficient photoreceptor integrity. ClinicalTrials.gov NCT01014052.

The retinal light response in animals originates from the photoisomerization of an opsin-coupled 11- cis -retinaldehyde chromophore. This visual chromophore is enzymatically produced through the action of carotenoid cleavage dioxygenases. Vertebrates require two carotenoid cleavage dioxygenases, β-carotene oxygenase 1 and retinal pigment epithelium 65 (RPE65), to form 11- cis -retinaldehyde from carotenoid substrates, whereas invertebrates such as insects use a single enzyme known as Neither Inactivation Nor Afterpotential B (NinaB). RPE65 and NinaB couple trans–cis isomerization with hydrolysis and oxygenation, respectively, but the mechanistic relationship of their isomerase activities remains unknown. Here we report the structure of NinaB, revealing details of its active site architecture and mode of membrane binding. Structure-guided mutagenesis studies identify a residue cluster deep within the NinaB substrate-binding cleft that controls its isomerization activity. Our data demonstrate that isomerization activity is mediated by distinct active site regions in NinaB and RPE65—an evolutionary convergence that deepens our understanding of visual system diversity. NinaB is an isomerooxygenase that generates visual chromophore (11- cis -retinal) from carotenoid substrates. Here Solano et al. reveal the structural basis for NinaB isomerase activity, providing new insights into the evolution of visual chromophore synthesis by carotenoid cleavage enzymes.

Lignin is the most abundant aromatic substrate on Earth and its valorization technologies are still under developed. Depolymerization and fragmentation are the predominant preparatory strategies for valorization of lignin to chemicals and fuels. However, due to the structural heterogeneity of lignin, depolymerization and fragmentation typically result in diverse product species, which require extensive separation and purification procedures to obtain target products. For lignin valorization, bacterial-based systems have attracted increasing attention because of their diverse metabolisms, which can be used to funnel multiple lignin-based compounds into specific target products. Here, recent advances in lignin valorization using bacteria are critically reviewed, including lignin-degrading bacteria that are able to degrade lignin and use lignin-associated aromatics, various associated metabolic pathways, and application of bacterial cultures for lignin valorization. This review will provide insight into the recent breakthroughs and future trends of lignin valorization based on bacterial systems.